1. Field of the Invention
This invention relates to a method for rapidly detecting the presence of duplex formation between single stranded nucleotide macromolecules, and more specifically, this invention relates to a method for using oligonucleotide arrays to rapidly detect duplex formation of oligonucleotide sequences. This invention also relates to a simple procedure for producing the oligonucleotide-arrays.
2. Background of the Invention
Present techniques for determining the existence of target sequences in patient DNA are complex, inefficient and somewhat time consuming. For example, one multi-step DNA sequencing approach, the Maxam and Gilbert method, involves first labeling DNA, and then splitting the DNA with a chemical, designed to alter a specific base, to produce a set of labeled fragments. The process is repeated by cleaving additional DNA with other chemicals specific for altering different bases, to produce additional sets of labeled fragments. The multiple fragment sets then must be run side-by-side in electrophoresis gels to determine base sequences.
Another sequencing method, the dideoxy procedure, based on Sanger, et al. Proc. Natl. Acad. Sci. USA 74, 5463-7 (1977) first requires the combination of a chain terminator as a limiting reagent, and then the use of polymerase to generate various length molecules, said molecules later to be compared on a gel. The accompanying lengthy electrophoresis procedures further detracts from the utility of this method as a fast and efficient sequencing tool.
A more recently developed sequencing strategy involves sequencing by hybridization on oligonucleotide microchips, or matrices, (SHOM) whereby DNA is hybridized with a complete set of oligonucleotides, which are first immobilized at fixed positions on a glass plate or polyacrylamide gel matrix. There are drawbacks to this technique, however. For instance, given that short nucleotide sequences are repeated rather frequently in long DNA molecules, the sequencing of lengthy genome strings is not feasible via SHOM. Also, hybridization with short oligonucleotides is affected by hairpin structures in DNA.
Furthermore, SHOM requires the utilization of high volume substrates containing many thousands of cells. If immobilized octamers are utilized to determine the positions of each of the four bases in genomic DNA, for example, then 4.sup.8 or 65,536 such octamers, themselves which would need to be previously fabricated, would have to be immobilized in individual cells on the gel matrix.
The production of literally thousands of these cells on the polyacrylamide substrates is problematic. First, these cells must be accurately spaced relative to one another. Second, these cells must be of sufficient depth and volume to hold predetermined amounts of the oligonucleotide. Cell sizes can range from 25 microns (.mu.m) to 1000 .mu.m.
Typically, cells are produced in a myriad of ways. Two-dimensional scribing techniques and laser evaporation are two typical methods of cell formation. Mechanical scribing techniques are limited, however, in that the smallest structures which can be produced via this method are approximately 100 .mu.m.times.100 .mu.m. Lasers applications, because of their expense, also are limiting. Furthermore, both of these procedures require complex equipment and experienced personnel.
A need exists in the art to provide a rapid and efficient method for detecting the existence of complementary sequences to target DNA strands. This detection method should be performed using standard reagents found in a typical biochemistry facility. A need also exists for a method to produce accurate polyacrylamide matrices to be used in the above-disclosed duplex detection method. Such a matrix production method also must be simple enough to be performed in typically-equipped biochemical laboratories.